Abstract
Oncogenic alterations in human epidermal growth factor receptor 2 (HER2) occur in approximately 2% of patients with non-small cell lung cancer and predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. Most clinical-grade tyrosine kinase inhibitors are limited by either insufficient selectivity against wild-type (WT) epidermal growth factor receptor (EGFR), which is a major cause of dose-limiting toxicity or by potency against HER2 exon 20 mutant variants. Here we report the discovery of covalent tyrosine kinase inhibitors that potently inhibit HER2 exon 20 mutants while sparing WT EGFR, which reduce tumor cell survival and proliferation in vitro and result in regressions in preclinical xenograft models of HER2 exon 20 mutant non-small cell lung cancer, concomitant with inhibition of downstream HER2 signaling. Our results suggest that HER2 exon 20 insertion-driven tumors can be effectively treated by a potent and highly selective HER2 inhibitor while sparing WT EGFR, paving the way for clinical translation.
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Data availability
CRISPR screening and expression data generated and analyzed in this study are deposited in the Gene Expression Omnibus with the accession code GSE181673. Structural data were deposited into the PDB with the accession code 7PCD. Source data have been provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
Code availability
Plots and statistical analyses were conducted with the statistical programming language R (v.3.5.0.). The code of the respective scripts is available upon request.
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Acknowledgements
We thank all colleagues at Boehringer Ingelheim RCV Oncology Research for discussions and critical feedback throughout the study. The project was funded by the Austrian Promotion Agency in 2019 with two grants within the general program (872827 and 0879012). We acknowledge Proteros Biostructures for their contributions to X-ray crystallography.
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Contributions
The medicinal chemistry sub-team was led by B.W. The biology sub-team was led by R.A.N. Design of inhibitors and synthetic routes was conducted by B.W., D.B., M.T., S.Z. and J.E.F. Inhibitor synthesis was performed by S.K., P.K., G.S., J. Bruchhaus, M.S., J. Balla, B.P.-S., J. Zimmer, S.M. and T.F. Selection of compounds for further profiling was carried out by B.W., D.S. and R.A.N. A. Bergner, D.B. and H.E. were responsible for the screening library. X-ray crystallography was the responsibility of G.B. The DMPK study design was conducted by D.S. The DMPK assays were performed by A. Beran. The biology sub-team consisted of A. Baum, V.S., P.C., R.S., D.A.B., C.R., A.H., A.K., M.C., S.L., L.L. and T.G. Setup of gene–drug screening pipeline was carried out by J. Zuber. Computational work and visualization of data was performed by D.G., M.B., A.S. and R.A.N. The study idea was conceived by B.W., P.E., M. Pearson, M. Petronczki, N.K., D.M., F.S. and R.A.N. The manuscript was written by B.W. and R.A.N.
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All authors except J. Zuber are or were full time employees of Boehringer Ingelheim.
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Nature Cancer thanks Ryohei Katayama, Antonio Passaro and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 DMPK parameters.
(a) Ba/F3 IC50 values in HER2YVMA vs. HER2YVMA,C805S cell lines. All results are quoted as the geometric mean ± SEM, pIC50 = -log IC50 (M). The number of repeats, n, are described in parentheses. (b) In vitro DMPK data for selected HER2YVMA inhibitors. PPB, plasma protein binding, solubility indicates kinetic solubility measured via an in-house HPLC method, ER, efflux ratio. n = 3 technical replicates. (c) In vivo mouse PK data for BI-1622 and BI-4142. Data shown are the geometric mean of n = 3 individual mice. MRT, mean residence time.
Extended Data Fig. 2 Structural information.
(a) Chemical structure of the HER2 TKI used as ligand in X-Ray crystallography in PDB 7PCD. (b) Ligand interaction diagram of the ligand shown in panel a with HER2. (c) Cellular IC50 data of the HER2 TKI used as ligand in the X-Ray crystal structure PDB 7PCD in comparison to BI-1622 and BI-4142. All results are quoted as the geometric mean ± SEM, pIC50 = -log IC50 (M). The number of repeats, n and the IC50, are described in parentheses. (d) X-Ray crystallography data collection, processing and refinement statistics.
Extended Data Fig. 3 Resistance and tumor cell data.
(a) N-ethyl-N-nitrosourea (ENU) resistance screen in Ba/F3 cells dependent on HER2YVMA. Compounds and concentrations as x-fold IC50 are indicated. Bar-plot indicates absolute count for number of times the resistance allele was observed. n = 2 for BI-4142, BI-1622, Afatinib and Poziotinib. (b) Left table: Cell proliferation assay in Ba/F3 cells (parental, or dependent on HER2YVMA or HER2YVMA,T862A) with indicated compounds. Right table: Cell proliferation assay in Ba/F3 cells (dependent on HER2YVMA or HER2YVMA,L800F) with indicated compounds. All results are quoted as the geometric mean ± SEM, pIC50 = -log IC50 (M). The number of repeats, n, and the IC50 are described in parentheses. (c) Confirmation of EGFR knockout and overexpression of exogenous EGFR variants by Western Blot in PC-9 cells. Representative image of two independent experiments. (d) Selectivity Index plot for proliferation assay in human tumor cells dependent on different oncogenic EGFR and HER2 variants in reference to EGFRWT dependent A431 cells. Number of n repeats is described in panel e of Extended Data Fig. 3. (e) Table summarizing the cell proliferation experiments in various tumor cell lines with indicated compounds. All results are quoted as the geometric mean ± SEM, pIC50 = -log IC50 (M). The number of repeats, n, and the IC50 are described in parentheses.
Extended Data Fig. 4 Biomarker modulation and gene expression changes.
(a) Heatmap visualizing gene expression profiling of pharmacodynamics biomarkers using BI-1622 and Poziotinib in NCI-H2170 HER2YVMA dependent cells sampling at 0.5 h, 2 h and 16 h (n = 3 independent experiments). Median gene expression values for each group are shown and z-score normalized across all samples with red colors represented high gene expression and blue colors showing low expression. (b) Reduction of pHER2 levels in NCI-H2170 HER2YVMA cells treated with increasing concentrations of BI-4142. Representative image of two independent experiments. (c) Dose response curves for phosphorylated HER2, phosphorylated ERK and DUSP6 mRNA levels. Representative image of two independent experiments. (d) Heatmap visualizing gene expression profiling of pharmacodynamics biomarkers using BI-4142 and Poziotinib in three cancer cell lines and sampling at 6 h and 24 h (n = 3 independent experiments). Median gene expression values for each group are shown and z-score normalized across all samples (n = 3) with red colors represented high gene expression and blue colors showing low expression.
Extended Data Fig. 5 Poziotinib efficacy and body weight data.
(a) Xenotransplantation experiment using NCI-H2170 HER2YVMA cells with Poziotinib at doses indicated in figure panel legend. Natrosol: n = 10 animals; Poziotinib 1 mg/kg qd n = 10 animals; Poziotinib 0.5 mg/kg bid n = 10 animals. The plot displays mean ± SEM. (b) Corresponding body weight changes for efficacy experiment shown in panel Extended Data Fig. 5a. The plot displays mean ± SEM.
Extended Data Fig. 6 Synthetic route for BI-1622 and BI-4142.
(a) Overview of the synthetic route to BI-1622. Reagents and conditions: i) i-PrOH, 45 °C, ii) mCPBA, DCM, 5 °C to r.t., iii) Boc-piperazine, DIPEA, DMF, 70 °C, iv) HCl in dioxane, DCM, methanol, r.t., v) acryloyl chloride, DCM, Et3N, 4 °C to r.t. (b) Overview of the synthetic route to BI-4142. Reagents and conditions: vi) Boc-piperazine, DIPEA, DMF, reflux, vii) oxalyl chloride, DCM, DMF, r.t., viii) i-PrOH, 45 °C, ix) HCl in dioxane, DCM, methanol, r.t., x) acrylic anhydride, DMAP, Et3N, DCM, r.t. or acryloyl chloride, DCM, Et3N, 4 °C to r.t.
Extended Data Fig. 7 1H NMR spectrum for BI-1622.
Graphical 1H NMR spectrum of BI-1622.
Extended Data Fig. 8 1H NMR spectrum BI-4142.
Graphical 1H NMR spectrum of BI-4142.
Supplementary information
Supplementary Information
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Wilding, B., Scharn, D., Böse, D. et al. Discovery of potent and selective HER2 inhibitors with efficacy against HER2 exon 20 insertion-driven tumors, which preserve wild-type EGFR signaling. Nat Cancer 3, 821–836 (2022). https://doi.org/10.1038/s43018-022-00412-y
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DOI: https://doi.org/10.1038/s43018-022-00412-y
